Indicated. The assay was started by addition of 0.8 mM Proto9 and

Indicated. The assay was started by addition of 0.8 mM Proto9 and 15900046 stopped after 5 min by adding fourFerrochelatase Refolding and KineticsFerrochelatase Refolding and KineticsFigure 6. Enzyme kinetic plots for FeCh and FeChD347 lacking His6-tags. 30 nM enzyme was analyzed in a continuous assay at 30uC. Hill equation fit relating initial rate (nM Zn-Proto9 s21) to Zn2+ concentration for FeChD347 (A), co-expressed FeCh (B) or refolded FeCh (C) to Zn2+ concentration. Michaelis-Menten equation fit was used when the variable Met-Enkephalin web substrate was Proto9 for FeChD347 (D), co-expressed FeCh (E) or refolded FeCh (F). Error bars represent standard deviation (n = 3). “Refolded FeCh” corresponds to in vitro folded enzyme, while “co-expressed FeCh” was coexpressed with chaperones assisting folding in E. coli cytosol. doi:10.1371/journal.pone.0055569.gvolumes of acetone. After centrifugation (13000 rpm for 10 min) the supernatant was transferred to a 2610 mm cuvette and ZnProto9 fluorescence was measured with excitation at 421 nm (slit width 3 nm) and emission spectra collected from 500 to 600 nm (slit width 5 nm).Results Expression, Purification and Refolding of His-FeCh from SynechocystisRefolding of proteins in vitro is dependent on various factors [30,31]. Recombinant ferrochelatase of the cyanobacterium Synechocystis 6803 was expressed in E. coli with an N-terminal His6-tag (His-FeCh) in inclusion bodies, and no soluble enzyme could be detected after immunoblotting (Fig. 2C, lane 2). Dialysis at 4uC overnight resulted in active enzyme, however, the sample still contained impurities, i.e. truncation products or soluble aggregates, that were problematic to remove irrespective of the addition of protease inhibitors (Complete from Roche Diagnostics or PMSF). We therefore developed a protocol to separate the Histagged full length protein with molecular mass of 47 kDa from other proteins by size exclusion chromatography under denaturing conditions (Fig. 2A) before refolding it on a Ni-IMAC column. This approach resulted in a yield of approximately 2 mg folded His-FeCh per liter culture in the absence of truncation products or soluble aggregates. An additive [30] had to be included to receive a substantial fraction of monomeric His-FeCh. In the presence of 0.5 M potassium chloride most protein passed through a 100 MWCO ultrafiltration membrane after refolding [30], and addition of 0.2 M MgCl2 or 1 glycine also improved monomerization (data not shown). To improve cryostability and refolding glycerol was added to the solution. Additionally, to solubilize the CAB-domain, which is predicted to form a transmembrane helix, sodium cholate detergent was used, as it has a small micellar size and passes through ultrafiltration membranes. The enzyme in its in vitro refolded state, was Tetracosactrin called “FeCh refolded”. The monomeric form of His-FeCh (and also FeCh) was separated from various oligomeric forms by size exclusion chromatography (Fig. 2D); no difference in activity could be observed between pure monomer and a mixture of monomeric and oligomeric proteins. In an attempt to purify soluble His-FeCh enzyme, different E. coli expression strains (Rosetta2, BL21, C41, Origami), growth temperatures (20uC, 30uC, 37uC) and IPTG concentrations (0.05 mM, 0.1 mM, 0.5 mM, 1 mM) were tested (data not shown). Additionally E. coli cells were stressed with 1 EtOH to force chaperone production and also a construct was created, in which maltose binding protein (MBP) was fused to the.Indicated. The assay was started by addition of 0.8 mM Proto9 and 15900046 stopped after 5 min by adding fourFerrochelatase Refolding and KineticsFerrochelatase Refolding and KineticsFigure 6. Enzyme kinetic plots for FeCh and FeChD347 lacking His6-tags. 30 nM enzyme was analyzed in a continuous assay at 30uC. Hill equation fit relating initial rate (nM Zn-Proto9 s21) to Zn2+ concentration for FeChD347 (A), co-expressed FeCh (B) or refolded FeCh (C) to Zn2+ concentration. Michaelis-Menten equation fit was used when the variable substrate was Proto9 for FeChD347 (D), co-expressed FeCh (E) or refolded FeCh (F). Error bars represent standard deviation (n = 3). “Refolded FeCh” corresponds to in vitro folded enzyme, while “co-expressed FeCh” was coexpressed with chaperones assisting folding in E. coli cytosol. doi:10.1371/journal.pone.0055569.gvolumes of acetone. After centrifugation (13000 rpm for 10 min) the supernatant was transferred to a 2610 mm cuvette and ZnProto9 fluorescence was measured with excitation at 421 nm (slit width 3 nm) and emission spectra collected from 500 to 600 nm (slit width 5 nm).Results Expression, Purification and Refolding of His-FeCh from SynechocystisRefolding of proteins in vitro is dependent on various factors [30,31]. Recombinant ferrochelatase of the cyanobacterium Synechocystis 6803 was expressed in E. coli with an N-terminal His6-tag (His-FeCh) in inclusion bodies, and no soluble enzyme could be detected after immunoblotting (Fig. 2C, lane 2). Dialysis at 4uC overnight resulted in active enzyme, however, the sample still contained impurities, i.e. truncation products or soluble aggregates, that were problematic to remove irrespective of the addition of protease inhibitors (Complete from Roche Diagnostics or PMSF). We therefore developed a protocol to separate the Histagged full length protein with molecular mass of 47 kDa from other proteins by size exclusion chromatography under denaturing conditions (Fig. 2A) before refolding it on a Ni-IMAC column. This approach resulted in a yield of approximately 2 mg folded His-FeCh per liter culture in the absence of truncation products or soluble aggregates. An additive [30] had to be included to receive a substantial fraction of monomeric His-FeCh. In the presence of 0.5 M potassium chloride most protein passed through a 100 MWCO ultrafiltration membrane after refolding [30], and addition of 0.2 M MgCl2 or 1 glycine also improved monomerization (data not shown). To improve cryostability and refolding glycerol was added to the solution. Additionally, to solubilize the CAB-domain, which is predicted to form a transmembrane helix, sodium cholate detergent was used, as it has a small micellar size and passes through ultrafiltration membranes. The enzyme in its in vitro refolded state, was called “FeCh refolded”. The monomeric form of His-FeCh (and also FeCh) was separated from various oligomeric forms by size exclusion chromatography (Fig. 2D); no difference in activity could be observed between pure monomer and a mixture of monomeric and oligomeric proteins. In an attempt to purify soluble His-FeCh enzyme, different E. coli expression strains (Rosetta2, BL21, C41, Origami), growth temperatures (20uC, 30uC, 37uC) and IPTG concentrations (0.05 mM, 0.1 mM, 0.5 mM, 1 mM) were tested (data not shown). Additionally E. coli cells were stressed with 1 EtOH to force chaperone production and also a construct was created, in which maltose binding protein (MBP) was fused to the.